Understanding how the directionality of vesicle traffic is maintained has been a long standing question in the field of membrane traffic. Recently, we have shown that the serine/threonine kinase Hrr25, a casein kinase I (CKI) family member, contributes to the directional delivery of ER-derived COPII coated vesicles to the Golgi. These studies have uncovered a previously unappreciated role for the importance of protein phosphorylation in ordering vesicle targeting, vesicle uncoating and fusion events. Hrr25/CKId is a member of a poorly understood, yet highly conserved, family of serine/threonine kinases that function in membrane traffic and other cellular processes. CKI family members were thought to be constitutively active kinases that regulate kinase-substrate interactions through subcellular localization and compartmentalization. Recently, however, we have found that the Rab GTPase Ypt1 (Rab1 in mammals) upregulates Hrr25 kinase activity. Rabs are molecular switches that cycle on and off membranes. They are recruited to membranes by guanine nucleotide exchange factors (GEFs) that activate the Rab at a specific location. The ability of Ypt1 to regulate the activity of Hrr25 ensures this kinase will only phosphorylate the vesicle-bound pool of the COPII coat. We propose four specific aims to address the role of CKI kinases in membrane traffic. These studies are likely to serve as a paradigm for the role of other CKI family members in membrane traffic. 1. We will use a variety of approaches (co-precipitation, in vitro phosphorylation assays, differential fractionation, column chromatography, in vitro transport and uncoating assays) to explore the possibility that the Rab GTPase Ypt1 regulates the uncoating of COPII vesicles via its ability to modulate the phosphorylation of a co-chaperone that is an Hrr25 substrate. 2. We will address if COPII and COPI vesicles are uncoated by a common mechanism. Specifically, we will ask if the same machinery that uncoats COPII coated vesicles also plays a role in disassembling the COPI cage from Golgi-derived vesicles. 3. We will ask if another CKI kinase family member, Yck3, can substitute for Hrr25 in ER-Golgi traffic. 4. We will address the role of other CKI kinase family members in membrane traffic.